ANTICANCER RESEARCH 36: 3289-3300 (2016)

Histone by ; A Classic DNA Methylating Anticancer Drug

TIELI WANG1, AMANDA J. PICKARD2 and JAMES M. GALLO2

1Department of and , California State University Dominguez Hills, Carson, CA, U.S.A.; 2Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, U.S.A.

Abstract. Background/Aim: The alkylating agent, adults (1). Despite advances in multimodal therapies of temozolomide (TMZ), is considered the standard-of-care for surgical extirpation, radiotherapy and chemotherapy, GBMs high-grade astrocytomas –known as multiforme remain a cancer of poor prognosis that can be attributed to (GBM)– an aggressive type of tumor with poor prognosis. The the aggressive nature of the tumors and the development of therapeutic benefit of TMZ is attributed to formation of DNA resistance to radiation and chemotherapy (2). Out of adducts involving the methylation of purine bases in DNA. We chemotherapeutic approaches to GBMs, temozolomide investigated the effects of TMZ on and amino (TMZ) is the main alkylating agent used, based on its ability acids, H3 and histone H3 . Materials to increase the median survival (3-6). and Methods: Chemical modification of amino acids, histone TMZ possesses favorable pharmacokinetic characteristics H3 and by TMZ was performed in phosphate with high oral bioavailability and blood-brain barrier (BBB) buffer at physiological pH. The reaction products were penetration (7). TMZ is a prodrug that undergoes pH- examined by mass spectrometry and western blot analysis. dependent degradation, first producing 5-(3-methyltriazen-1- Results: Our results showed that TMZ following conversion to yl)imidazole-4-carboxamide (MTIC), that is subsequently a methylating cation, can methylate histone H3 peptide and degraded into 4-amino-5-immidazole-carboxamide (AIC) – an histone H3 protein, suggesting that TMZ exerts its anticancer inactive metabolite and a methyldiazonium cation, the activity not only through its interaction with DNA, but also methylating agent (Figure 1). The anticancer effects of TMZ through alterations of protein post-translational modifications. are attributed to the methylation of DNA by TMZ with the Conclusion: The possibility that TMZ can methylate primary cytotoxic lesion being O6-methylguanine (O6-MeG); involved with epigenetic regulation of protein indicates a N7-methylguanine, N3-methylguanine, and N3-methyladenine potentially unique mechanism of action. The study will (8-10) adducts also contribute to the cytotoxicity. The DNA contribute to the understanding the anticancer activity of TMZ adducts can lead to single- and double-strand DNA breaks and in order to develop novel targeted molecular strategies to eventually apoptosis; however, numerous DNA repair systems, advance the cancer treatment. highlighted by the methylguanine (MGMT) that repairs the O6-MeG lesions, can result in Malignant , or glioblastoma multiforme (GBM), are chemoresistance (11-13). Other DNA repair processes, such as the most common primary brain malignancies found in mismatch repair (MMR) and base excision repair (BER), also influence cellular resistance to TMZ. The field of characterizes how chemical and enzymatic modifications to DNA and histones occur and This article is freely accessible online. their subsequent impact on expression. In fact, Correspondence to: Tieli Wang, Department of Chemistry and hypermethylation of the MGMT in GBM patients Biochemistry, California State University Dominguez Hills, Carson, (14, 15, 16) is used as a favorable prognostic biomarker and CA, U.S.A. Tel: 310 2433388, Fax: 310 2432593, e-mail: increases drug sensitivity since a lower amount of the [email protected] and James M. Gallo, Department of enzyme is available to repair TMZ-induced lesions (17). Pharmacology and Systems Therapeutics, Icahn School of Medicine Although DNA methylation has been shown to affect at Mount Sinai, New York, NY, U.S.A. Tel: 212 2413980, Fax: 212 structure and influence (18), it 9967214, e-mail: [email protected]. is direct enzymatic modification of histones, such as Key Words: Temozolomide, malignant gliomas, chemotherapy, methylation and that are considered crucial to histone methylation. .

0250-7005/2016 $2.00+.40 3289 ANTICANCER RESEARCH 36: 3289-3300 (2016)

Figure 1. TMZ metabolic cascade at physiological pH to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) and its metabolites, 4-amino-5- imidazole-carboxamide (AIC) and methyldiazonium ion.

In the current study, we addressed whether TMZ can reaction mixture was centrifuged in an Amicon Ultra-0.5 centrifugal methylate histones directly, by examining the effects of TMZ filter device (EMD Millipore, Billerica, MA, USA) at 4˚C at 14,000 × on amino acids, peptides and histone proteins in vitro using g with a 40˚ fixed angle rotor to concentrate the protein samples and remove low molecular weight impurities and unreacted TMZ. The 100 mass spectrometry and protein biochemical methods. Our μg concentrated protein solution was resuspended in 100 μl 0.01 M results demonstrated that TMZ is able to methylate histone phosphate buffer (pH 7.4). Western blot analysis was then used to proteins under physiological conditions. The possibility that confirm the methylated histone H3 protein. this action could occur in GBM patients opens a new avenue of investigation to determine its significance. Mass spectrometry. Mass spectrometry experiments were performed using a QTRAP 5500 triple quadrupole mass spectrometer (AB Materials and Methods SCIEX, Framingham, MA, USA) coupled to a Shimadzu Prominence UFLC system (Shimadzu Corp., Torrance, CA, USA). The system was continuously supplied with ultra-high purity Chemicals and general procedures. Arginine, lysine and TMZ , dry air, and zero air by a Source 5000 LC-MS TriGas (>98%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, generator (Parker Hannifin Corp., Haverhill, MA, USA). The USA). Histone H3 (1-8) peptide H2N-ARTKQTAR-COOH (>95% QTRAP 5500 system was equipped with an electrospray ionization by HPLC, MW=931.1 g/mol) was purchased from AnaSpec, Inc. (ESI) TurboIon Spray probe utilizing zero grade air as the nebulizer (Fremont, CA, USA). Xenopus recombinant histone H3 (C110A) (Gas 1) and heater (Gas 2) gases. Ultra-high purity nitrogen was (>98% by SDS-PAGE, MW=15,239 Da), supplied as a lyophilized used as the curtain (CUR) and collision (CAD) gases. Mass powder, was purchased from Active Motif (Carlsbad, CA, USA). spectrometer control and spectral processing were carried out using Recombinant human histone H3.1 protein (MW=15273.2 Da by Analyst 1.5.2 software (AB SCIEX). Mass spectrometer parameters ESI-TOF) was purchased from New England BioLabs, Inc. were optimized for each compound analyzed (Tables I and II). A (Ipswich, MA, USA). Human recombinant histone H3 protein 1μl sample of unreacted , TMZ, or the reaction mixture (≥95% by SDS-PAGE, MW=15,500 Da by SDS-PAGE) was was introduced into the electrospray source by direct injection. The purchased from Cayman Chemical Co. (Ann Arbor, MI, USA) as a mobile phase consisted of 50% methanol with 0.1% formic acid and lyophilized powder. All buffers and mobile phases were prepared 50% with 0.1% formic acid and was maintained at a constant using analytical grade reagents and ultrapure water from a Millipore flow rate of 200 μl/min. Unreacted and reacted peptide solutions Synergy UV water filtration system (EMD Millipore, Billerica, MA, were infused directly into the electrospray source at a flow rate of 7 USA) at a resistivity of 18 MΩ·cm. All reagents were used without μl/min. All mass spectra were acquired in positive-ion mode with further purification. The pH measurements were measured using a unit mass resolution. Mass spectra for the free amino acid studies calibrated Fisher Scientific AccuMet Basic AB15 pH MV benchtop were background-subtracted with a spectrum obtained of 100 μM pH meter (Fisher Scientific, Pittsburgh, PA, USA). TMZ in 50/50 0.01 M phosphate buffer/methanol (v/v). Peptides were fragmented under conditions with the collision Reactivity of amino acids and peptides with TMZ. Separate stock energy between 15 to 45 eV by the direct infusion method at a flow solutions of arginine (0.5 mM), lysine (0.5 mM), TMZ (0.2 mM) rate of 7 μl/min. The MS/MS spectra were interpreted manually, and histone H3 (1-8) peptide (10 mg/ml) were prepared in 0.01 M primarily by assigning the peaks corresponding to the C-terminal y- phosphate buffer (pH 7.4) and kept at 4˚C until use. A solution ions. The modification was mapped based on the mass difference of containing 10 μM amino acid or peptide and 200 μM TMZ was the y ions in the spectrum derived from the doubly charged mixed at room temperature for 24 h. Subsequently an aliquot of the precursor ions when compared to the y ions in the spectrum of solution was diluted 1:1 with methanol and was passed through a unmethylated peptide. Mass spectrometer parameters were 0.22 μm syringe filter. The filtered solution was subsequently optimized for each methylated peptide analyzed (Table III). Mass analyzed by LC-MS/MS. fragmentation prediction was generated using Mascot (Matrix Science, London, UK). Reactivity of histone H3 Proteins with TMZ. 100 μg of histone H3 Western blot analysis. Standard protein electrophoresis conditions and protein was resuspended in 100 μl 0.01 M phosphate buffer (pH 7.4) reagents were used for gel and sample preparation. Recombinant and 10 μM TMZ in 0.01 M (pH 7.4) phosphate buffer was mixed with proteins (0.5-5 μg) and the LI-COR Chameleon Duo ladder (LI-COR the protein solution at room temperature for 24 h. The resultant Biosciences, Inc., Lincoln, NE) were loaded onto a 12% Mini-Protean-

3290 Wang et al: by Temozolomide

Table I. Optimized mass spectrometer parameters for tandem mass spectrometry of arginine and methylated arginine.

Q1 CAD CUR DP TEM IS GS1 GS2 EP CE CXP (Da) (psi) (V) (˚C) (V) (psi) (psi) (V) (V) (V)

R 174.600 MED 20 50 550 4500 60 60 10 20 12 MMR 189.100 MED 20 70 550 5500 60 60 10 20 11 DMR 203.200 MED 10 60 450 5500 60 60 10 20 11 TMR 217.100 MED 10 30 500 5500 60 60 11 25 11

R, Arginine; MMR, monomethylarginine; DMR, dimethylarginine; TMR, trimethylarginine.

Table II. Optimized mass spectrometer parameters for tandem mass spectrometry of lysine and methylated lysine.

Q1 CAD CUR DP TEM IS GS1 GS2 EP CE CXP (Da) (psi) (V) (˚C) (V) (psi) (psi) (V) (V) (V)

K 147.000 MED 20 50 550 4500 60 50 10 18 12 MMK 161.000 LOW 20 66 600 4000 60 60 10 18 12 DMK 175.000 LOW 20 66 600 4000 60 60 10 20 12 TMK 189.000 LOW 20 66 600 4000 60 60 10 25 12

K, Lysine; MMK, monomethylysine; DMK, dimethylysine; TMK, trimethylysine.

TGX precast gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Table III. Optimized mass spectrometer parameters for Q1 scans of Electrophoresis was carried out in 1X Tris//SDS gel TMZ reacted with arginine (R + TMZ), lysine (K + TMZ), peptide (PEP electrophoresis buffer (Bio-Rad, Hercules, CA) for 1 h at 100 V + TMZ) and peptide alone (PEP). according to the procedure recommended by the manufacturer. Proteins were then transferred overnight to a PVDF membrane pre- CUR DP TEM IS GS1 GS2 EP wet with methanol and transfer buffer (1X Tris/Glycine). The (psi) (V) (˚C) (V) (psi) (psi) (V) membrane was blotted with Odyssey blocking buffer (TBS) for 1 h, followed by monoclonal Anti-β- antibody produced in mouse R + TMZ 20 66 500 5500 50 50 10 K + TMZ 20 66 500 5500 50 50 10 (Sigma-Aldrich Co., St. Louis, MO) and polyclonal anti-histone PEP 20 66 450 4500 40 60 11 antibody produced in rabbit (Active Motif, Carlsbad, CA) PEP + TMZ 20 66 450 4500 40 60 11 that was diluted 1:1000 in Odyssey blocking buffer plus 0.2% Tween- 20 for 3 h. Following standard washing procedure, the membrane was then blotted with IRDye® 800CW Goat anti-Rabbit IgG (H+L) and IRDye® 680RD Goat anti-Mouse IgG (H+L) (LI-COR Biosciences, Inc., Lincoln, NE) diluted 1:10,000 in Odyssey blocking buffer plus 0.2% Tween-20 for 1 hour in the dark. The membrane was washed Nα,Nα-dimethylarginine (Nα,Nα -DMR), and Nα,Nα, Nα- three times with TBS plus 0.1% Tween-20 and imaged by near- trimethylarginine (Nα,Nα, Nα -TMR) (21), and serve as a infrared fluorescence detection on a LI-COR Odyssey CLx scanner basis to compare to methylated species produced by TMZ. (LI-COR Biosciences, Inc., Lincoln, NE). The reaction of TMZ with arginine resulted in a mixture of mono-, di- and tri-methylated products that were detected at Results m/z 190, 204 and 218, respectively, with a 14 Da increase in the mass for each subsequent methylation (Figure 2B). The Methylation of arginine and lysine by TMZ. Experimental methylation positions could be either at Nα or at Nε position. and natural methylation sites of arginine and lysine (Figure The identities of the methylated products were further 2A) can occur on the side-chain nitrogen atoms, both investigated by the collision-activated dissociation (CID) symmetrically and asymmetrically in arginine (19), and less mass fragmentation pattern to determine the potential often at the N-terminal nitrogen atoms and nitrogen methylation sites. The CID patterns of arginine and its atoms in peptide bonds (20). Methylation products of methylated products displayed similar characteristic arginine reacted with formaldehyde or methyl iodide have fragmentation patterns to the Nα-MMR, Nα,Nα -DMR, and been described as Nα-monomethylarginine (Nα -MMR), Nα,Nα, Nα-TMR products as described previously,

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Figure 2. (a) Potential methylation sites of arginine and lysine free amino acids. Mass spectra of the reaction between TMZ and (b) arginine or (c) lysine demonstrating multiple .

suggesting that TMZ may methylate the N-terminal nitrogen characterized as Nε- (Nε-MMK), Nε,Nε- atoms of arginine or the nitrogen atoms of arginine side dimethyllysine (Nε,Nε- DMK), and Nε,Nε,Nε-trimethyllysine chains (Figure 3). (Nε,Nε,Nε-TMK) (21). The reactions between TMZ and lysine were analogous to those with arginine and produced a mixture of mono-, di-, Mapping the methylation site in a histone peptide following and tri-methylated lysine products at m/z 148, 162, and 176, the TMZ treatment. When methyl groups (-CH3) replace respectively, with a 14-Da increase in the mass for each atoms on a peptide, the isotopic mass gains are 14 subsequent methylation (Figure 2C). CID fragmentation Da for each methyl substitution. The substitution can occur patterns of the TMZ-induced methylation products (Figure at the amino and carboxyl termini and at the side chains of 4) suggest N-methylation products previously lysine and arginine (22). The b and y ions produce the

3292 Wang et al: Protein Methylation by Temozolomide nine (DMR) and (D) protonated trimethylated nine (DMR) and (D) protonated ESI/CID mass spectra of (A) protonated arginine, (B) protonated monomethylated arginine (MMR), (C) protonated dimethylated argi (MMR), (C) protonated monomethylated arginine (B) protonated arginine, ESI/CID mass spectra of (A) protonated arginine (TMR). arginine Figure 3.

3293 ANTICANCER RESEARCH 36: 3289-3300 (2016) DMK) and (D) protonated trimethylated lysine DMK) and (D) protonated ESI/CID mass spectra of (A) protonated lysine, (B) protonated monomethylated lysine (MMK), (C) protonated dimethylated lysine ( monomethylated lysine (MMK), (C) protonated lysine, (B) protonated ESI/CID mass spectra of (A) protonated (TMK). Figure 4.

3294 Wang et al: Protein Methylation by Temozolomide

Figure 5. Fragmentation map of human histone H3 peptide 1-8 Figure 6. Fragmentation map of methylated human histone H3 peptide (ARTKQTAR). A. The ladder or family of “b ions” and “y ions” 1-8 (ARTKQTAR-me). (me) is on 8 (arginine). generated after that may be observed in the fragment mass When methylation occurs on the amino acid, the m/z of b ion or y ion spectrum for this peptide. Y1 represents spectra for fragmentation of H3 will display the shift based upon the position of amino acid in the peptide on R175, Y2 represents spectra for fragmentation of H3 peptide peptide and molecular weight of the attachment. Y1 represents spectra on R246, etc. B1 represents spectra for fragmentation of H3 peptide on for fragmentation of H3 peptide on R189, Y2 represents spectra for A72, b2 represents spectra for fragmentation of H3 peptide on A 228, etc. fragmentation of H3 peptide on R260, etc. B1 represents spectra for B. Fully annotated fragmentation nomenclature after ion fragmentation of H3 peptide on A72, b2 represents spectra for calculator results. The blue lines represent the fragmentation of H3 fragmentation of H3 peptide on A228, etc. B. Fully annotated peptide at the corresponding peptide bond for each amino acid. B- and y- fragmentation nomenclature after bond cleavage between amino acids. fragment ions are denoted by blue and red, respectively. The blue lines represent the fragmentation of H3 peptide on amino acid on each peptide bond.

characteristic footprint necessary for the identification of predicted b and y ions for the unmethylated peptide, while possible methylation sites, which are generated by the the table in Figure 7C lists the m/z for the predicted b and fragmentation of the peptide bonds between two adjacent y ions for the methylated peptide at the Arg side chain at amino acid residues. Those fragment peaks that appear to the C-terminus. The methylation was sufficiently stable extend from the amino terminus are termed “b ions”, under CID conditions that resulted in cleavage of the whereas groups of peptide fragment ions that appear to peptide backbone wherein it could be located in the extend from the carboxyl terminus are termed “y ions” sequence of the peptide by the observed y ions. Mapping (Figures 5 and 6). the detailed methylation sites for multi-methylated peptides The reaction products of TMZ with human histone H3 was not as conclusive as for MMP (data not shown). The peptide 1-8 (ARTKQTAR) were identified as the mono- result demonstrated that TMZ is able to methylate amino methylated peptide (MMP), tri-methylated peptide (TMP), acid side chains in the peptide. tetra-methylated peptide (TetraMP), hexa-methylated peptide (HexaMP), hepta-methylated peptide (HeptaMP), Methylation of histone H3.1 protein by TMZ. To octa-methylated peptide (OctaMP), nona-methylated demonstrate that TMZ, via its methyldizaonium cation, can peptide (NonaMP) and deca-methylated peptide (DecaMP) methylate arginine and lysine within a protein, the Xenopus at m/z 946.6, 974.4, m/z 990.1, m/z 1012.6, m/z 1030.1, and human histone H3.1 recombinant proteins were reacted m/z 1041.7, m/z 1055.3, and m/z 1072.3, respectively with TMZ. Methylation of the xenopus histone H3.1 was (Figure 7A). The methylation site for MMP was mapped observed at a higher molecular weight than unmethylated using low-energy CID fragmentation (45 eV) (23). In the protein (data not shown), as determined by gel spectrum recorded from the unmethylated histone peptide electrophoresis. Methylated human histone H3.1 displayed (Figure 7B), the b ions and y ions in the spectrum derived a similar molecular shift (data not shown). Western blot from the doubly-charged precursor ions were displayed. analysis was then used to further confirm the methylation The MS/MS spectrum of MMP shown in Figure 3C status of the proteins. As shown in Figure 8, both xenopus displayed a 14 Da shift for all y ions in comparison with and human histone H3.1 proteins are positive for western the y ions in the spectrum of unmodified peptide. The blot analysis using H3K4Me antibody that is specific for the methylation site was identified on number 8 arginine methylation of the lysine residue at the N-terminal number residue according to the mass fragmentation prediction 4 position. Western blot analysis support that both Xenopus from Mascot shown in the table above the spectra in figure and human histone H3.1 proteins are methylated by TMZ 3B and 3C. The table in Figure 7B shows the m/z of the under physiological conditions.

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Figure 7. (a) Mass spectrum of histone H3 peptide (1-8) after reacting with TMZ. Abbreviations: [P+H]+, native peptide; MMP, mono-methlated peptide; TMP, tri-methylated peptide; TetraMP, tetra-methylated peptide; HexaMP, hexa-methylated peptide; HeptaMP, hepta-methylated peptide; OctaMP, octa-methylated peptide; NonaMP, nona-methylated peptide; DecaMP, deca-methylated peptide. (b) Fragmentation of the doubly-charged precursor ion corresponding to the unmethylated histone H3 peptide. (c) Fragmentation of the doubly-charged precursor ion corresponding to MMP.

Discussion Emerging ideas in systems pharmacology beckon for a detailed molecular analysis of drug action to ensure that all TMZ is a clinically important methylating agent used in drug targets are identified (24, 25). Such a comprehensive chemotherapy against malignant gliomas whose efficacy is understanding of the mechanisms of drug action should limited by the development of drug resistance (11). provide greater insight into why drugs succeed or fail in

3296 Wang et al: Protein Methylation by Temozolomide

Figure 8. Western blot analysis of histone H3.1 proteins; 1 μg methylated Xenopus histone H3.1 (Lane 1), 10 μg methylated human histone H3.1 (Lane 2), 0.5 μg methylated human histone H3.1 (Lane 3), 2 μg methylated human histone H3.1(Lane 4), 1 μg Xenopus histone H3.1 (Lane 5), 10 μg Human histone 3.1 (Lane 6), 0.5 μg human histone H3.1 (Lane 7), and 2 μg human histone H3.1 (Lane 8).

patients. The current results indicating the ability of TMZ to glutathionylation, and ubiquitylation, protein methylate histone proteins suggest another target that may methylation and play an important role in be involved in its mechanism of action. This is an intriguing turning on and off certain cellular signaling pathways. When finding since histone modifications are the basis of methyl groups replace hydrogen atoms at the side-chain of epigenetic modifications critical to gene . lysine and arginine residues, it will alter the structure of the Numerous anticancer drug discovery efforts are directed at protein they bind, hydrophobicity and the size of the histone including methylating and demethylating group in the side chain of lysine and arginine residues. As a reactions and complexes involved in histone remodeling. The result, the electrostatic forces governing the histone-DNA possibility that TMZ could serve as a histone methylating interaction will be reduced and the gene expression of the agent offers a new avenue for exploration. DNA will be affected. Methylation can also change the Methylation is a well-known mechanism of epigenetic hydrophobicity of the vicinity of protein to activate and regulation. Alteration of histone methylation status deactivate different portions of chromatin by blocking influences the availability of DNA for transcriptional transcription factors and other proteins from accessing the regulation, DNA repair, RNA processing and signal DNA. This is an important regulatory mechanism that allows transduction (26). Histone H3 is an important component of different cells to express different portions of the genome. It the core that forms , which are is likely that methylation of protein can contribute to the the basic repeating units of chromatin. Chromatin dynamic control of biological processes. organization plays an important role in gene-expression Global levels of histone modifications differ between cell regulation through conformational changes from an open to types and they have been found to be associated with the a closed configuration or vice versa. The core histone clinical outcome and progression of different cancer types. proteins are subjected to a variety of post-translational Low levels of dimethylation of histone lysine number 4 residue modifications in both their random coiled N-terminal tails (H3K4me2) correlated with low survival rates in both lung and and their globular central cores. One of those post- kidney cancers (27). Low levels of H3K4me2 also correlated translational modifications involves methylation of lysine with adverse prognosis in non-small cell lung carcinomas and arginine residues (26). Like , (NSCLC), hepatocellular carcinomas (HCC) and breast cancers

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(28). On the other hand, an increase in dimethylation states of 7 Bellesta A, Zhou Q, Zhang X, Lv H and Gallo JM: CPT: H3K4me2 was found in pancreatic cancer, adenocarcinoma, Pharmacometrics Syst Pharmacol 3: 1-11, 2014. hormone factory prostate cancer (HRPC) compared to 8 Wedge SR and Newlands ES: O6-benzylguanine enhances the clinically localized cancers (29, 30). Histone modification can sensitivity of a lioma xenograft with low O6-alkyguanine-DNA alkytransferase activity to temozolomide and BCNU. 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24 Tentori L and Graziani G: Recent approaches to improve the 29 Watanabe T, Mornaga S, Akaike M, Numata M, Tamagawa H, antitumor efficacy of temozolomide. Curr Med Chem 16: 245- Yamamoto N, Shiozawa M, Kameda Y, Nakamura Y and Miyagi 257, 2009. Y: The cellular level of histone H3 lysine dimethylation 25 Zhang J, Stevens MF and Bradshaw TD: Temozolomide: correlates with response to adjuvant gemcitabine in Japanese Mechanisms of Action, Repair and Resistance. Curr Mol pancreatic cancer patients treated with surgery. Euro J Surg Pharmacol 5: 102-104, 2012. Oncol 38: 1051-1057, 2012. 26 Berger SL: The complex language of chromatin regulation 30 Manuyakorn A, Paulus R, Farrell J, Dawson NA, Tze S, during transcription. Nature Method 447: 407-412, 2007. Cheung-Lau G, Hines OJ, Reber H, Seligson DB, Horvath S, 27 Seligson DB, Horvath S, McBrian MA, Mah V, Yu, H, Tze S, Kurdistani SK, Guha C and Dawson DW: Cellular histone Wang Q, Chia D, Goodglick L and Kurdistani SK: Global levels modification patterns predict prognosis and treatment response of histone modifications predict prognosis in different cancers. in resectable pancreatic adenocarsinoma: results from RTOG Am J Pathol 174: 1619-1628, 2009. 9704. J Clin Oncol 28: 1358-1365, 2010. 28 Magerl C, Ellinger J, Braunschweig T, Kremmer E, Koch LK, Holler T, Buttner R, Luscher B and Gutgemann I: H3K4 dimethylation in hepatocellular carcinoma is rare compared with other hepatobiliary and gastrointestinal carcinomas and Received April 1, 2016 correlates with expression of the methylase Ash2 and the Revised June 6, 2016 LSD1. Hum Pathol 41: 181-189, 2010. Accepted June 8, 2016

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